U.S. patent application number 10/713898 was filed with the patent office on 2006-04-27 for micro fluidic system for single molecule imaging.
Invention is credited to Juan J. de Pablo, Eileen T. Dimalanta, David C. Schwartz.
Application Number | 20060088944 10/713898 |
Document ID | / |
Family ID | 36206673 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060088944 |
Kind Code |
A1 |
Schwartz; David C. ; et
al. |
April 27, 2006 |
Micro fluidic system for single molecule imaging
Abstract
Laminar flow of a carrier liquid and polymeric molecules through
micro-channels is used to straighten the polymeric molecules and
attach the straightened molecules to a wall of the micro-channel
for subsequent treatment and analysis. Micro-channels can be
manufactured using an elastic molding material. One micro-channel
embodiment provides fluid flow using a standard laboratory
centrifuge.
Inventors: |
Schwartz; David C.;
(Madison, WI) ; Dimalanta; Eileen T.; (Madison,
WI) ; de Pablo; Juan J.; (Madison, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE, SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
36206673 |
Appl. No.: |
10/713898 |
Filed: |
October 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09962802 |
Sep 24, 2001 |
6610256 |
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10713898 |
Oct 18, 2002 |
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08855410 |
May 13, 1997 |
6294136 |
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09962802 |
Sep 24, 2001 |
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08415710 |
Apr 3, 1995 |
5720928 |
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08855410 |
May 13, 1997 |
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Current U.S.
Class: |
436/174 ;
264/219; 264/435; 422/400; 435/6.1 |
Current CPC
Class: |
G01N 1/30 20130101; G01N
1/40 20130101; Y10T 436/25 20150115; G01N 1/405 20130101 |
Class at
Publication: |
436/174 ;
264/435; 264/219; 422/100; 435/006 |
International
Class: |
H05B 6/00 20060101
H05B006/00; B29C 33/40 20060101 B29C033/40; G01N 1/10 20060101
G01N001/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with United States government
support awarded by the following agencies: DOE DE-FGO2-99ER62830
and NIH HG00225.
[0002] The United States has certain rights in this invention.
Claims
1. An apparatus for fixing and straightening polymeric molecules,
the apparatus comprising: a micro-channel sized to provide laminar
flow of a liquid along a micro-channel length, wherein at least a
first wall of the micro-channel provides electrostatic attraction
to the polymeric molecule; means for passing the liquid and
polymeric molecule through the micro-channel operating together
with the micro-channel to: (a) straighten the polymeric molecule by
passage along the micro-channel within the laminar flow, and (b)
cause adsorption of the polymeric molecule to the first wall of the
micro-channel in straightened form.
2. The apparatus of claim 1 wherein the first wall of the
micro-channel is transparent.
3. The apparatus of claim 1 wherein the first wall of the
micro-channel is glass.
4. The apparatus of claim 1 wherein the first wall is treated to
have a positive surface charge of predetermined density.
5. The apparatus of claim 1 including further at least one second
wall of the micro-channel provides less electrostatic attraction to
the polymeric molecule than the first wall.
6. The apparatus of claim 1 wherein the first wall is detachable
from the micro-channel.
7. The apparatus of claim 1 wherein the micro-channel is formed at
least in part from poly(dimethylsiloxane).
8. The apparatus of claim 1 wherein the micro-channel size and a
rate of flow of the liquid and polymeric molecule is selected so
that diffusion of the polymeric molecule dominates sedimentation of
the polymeric molecule.
9. The apparatus of claim 1 wherein a width of the micro-channels
is between 1 and 0.01 times the straightened length of the
polymeric molecule.
10. The apparatus of claim 1 wherein a width of the micro-channel
is less than the diffusion distance of an end of the polymeric
molecule during a passage time through the micro-channel.
11. The apparatus of claim 1 wherein a width of the micro-channel
is less than 100 micrometers.
12. The apparatus of claim 1 wherein the liquid has a viscosity
substantially equal to the viscosity of water.
13. The apparatus of claim 1 wherein the adsorption of the
straightened polymeric molecule is preceded by attachment of at
least one end of the polymeric molecule to the first wall.
14. The apparatus of claim 1 wherein at least one end of the
micro-channel provides a funnel section opening to a reservoir
holding the liquid and polymeric molecules.
15. The apparatus of claim 1 wherein the means for passing the
liquid and polymeric molecule through the micro-channel is a
positive pressure pump attached at one end of the
micro-channel.
16. The apparatus of claim 1 wherein the means for passing the
liquid and polymeric molecule through the micro-channel is a
negative pressure pump attached at one end of the
micro-channel.
17. The apparatus of claim 1 wherein the means for passing the
liquid and polymeric molecule through the micro-channel is a
reservoir acted on by a force resulting from centrifugal
acceleration.
18. The apparatus of claim 17 wherein the reservoir is at least end
well extending perpendicular to the length of the micro-channel and
wherein the apparatus further includes a housing allowing the end
well and micro-channel to be received by a centrifuge with the end
well extending along a principal axis of centrifugal acceleration
and the micro-channel extending substantially across the principal
axis of centrifugal acceleration.
19. The apparatus of claim 1 wherein the polymeric molecule is
DNA.
20. The apparatus of claim 1 wherein the micro-channel includes a
region of varying cross section to promote a gradient in flow
rate.
21. A method of straightening and fixing polymeric molecules
comprising the steps of: (a) putting the polymeric molecules in a
carrier liquid (b) passing the polymeric molecules and carrier
liquid through a micro-channel having a first wall
electrostatically attractive to the polymeric molecule to promote a
laminar flow of carrier liquid in the micro-channel causing the
polymeric molecule to adhere in straightened configuration to the
first wall.
22. The method of claim 21 further including the step of (c)
detaching the first wall from the micro-channel.
23. The method of claim 21 further including the step of (c)
applying restricting enzymes to the straightened polymeric molecule
attached to the first wall.
24. The method of claim 21 further including the step of (c)
optically inspecting the straightened polymeric molecule at
attached to the first wall.
25. The method of claim 21 further wherein step (b) first causes a
straightening of the polymeric molecule in the laminar flow and
second causes attachment of one end of the polymeric molecule to
the first wall and third causes attachment of the length of the
polymeric molecule to the wall.
26. The method of claim 21 wherein the polymeric molecules are
treated with a condensation agent to collapse the polymeric
molecules into shear resistant balls and wherein step (a) includes
the step of placing the polymeric molecules and carrier liquid into
a reservoir attached to the micro-channel and decondensing the
polymeric molecules in the reservoir prior to step (b).
27. The method of claim 21 further including the step of treating
at least one wall of the micro-channel to have a positive surface
charge of predetermined density.
28. A method of manufacturing a micro-channel for straightening and
fixing polymeric molecules, comprising the steps of" (a) preparing
a mold of the micro-channels having a base plate and upstanding
micro-channel cores (b) coating the mold with an elastic molding
compound (c) removing the cured elastic molding compound from the
mold; (d) applying the cured elastic molding compound to an optical
mapping surface to create micro-channels between the molding
compound and the optical mapping surface.
29. The method of claim 28 including the step of treating the
optical mapping surface to have a positive surface charge of
predetermined density.
30. The method of claim 28 including the step of wherein the
optical mapping surface is transparent.
31. The method of claim 28 including the step of wherein the
optical mapping surface is glass.
32. The method of claim 28 including the step of wherein the
elastic compound is poly(dimethylsiloxane).
33. An apparatus for fixing and straightening polymeric molecules,
the apparatus comprising: a micro-channel sized to provide laminar
flow of a liquid along a micro-channel length including a first
wall of the micro-channel providing electrostatic attraction to the
polymeric molecule; means for passing the liquid and polymeric
molecule through the micro-channel operating together with the
micro-channel to: (a) straighten the polymeric molecule by passage
along the micro-channel within the laminar flow, and (b) cause
adsorption of the polymeric molecule to the first wall of the
micro-channel in straightened form; wherein the first wall of the
micro-channel is releasably attached to remaining walls of the
micro-channel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
BACKGROUND OF THE INVENTION
[0003] The present invention relates to methods of manipulating
molecules and, in particular, to a fluid transport system useful
for straightening, aligning, and fixing long chain polymers such as
DNA.
[0004] The analysis of nucleic acid molecules (e.g. DNA) and, in
particular, the sequencing of such molecules may be aided by
optical techniques in which long portions of such molecules are
straightened and fixed to a substrate for microscopic analysis. The
fixed molecule may be analyzed by creating "landmarks" on the
molecule by attaching fluorescent markers to specific locations or
by cutting it with restriction enzymes to form visible breaks at
specific locations. The order and relative separation of the
landmarks is preserved, because the molecule remains fixed, and may
be used to produce an optical map of the molecule. The optical map
provides a framework on which other sequence information may be
assembled. The landmarks allow optical maps of fragments of long
molecules to be assembled into the entire molecule by the process
of matching fragments with similarly spaced landmarks.
[0005] The effective use of optical maps requires that large
numbers of single molecules be processed. A number of techniques
have been examined for the purpose of straightening and fixing
large numbers of molecules including: (1) straightening the
molecules in a flow of molten gel which is then hardened to fix the
molecules in place and (2) straightening the molecules under
capillary flow of a carrier liquid or convective flow caused by
evaporation of a carrier liquid and promoting adsorption of the
elongated molecules to a substrate adjacent to the flow.
[0006] A different set of techniques has been investigated in which
the molecules are straightened in a flowing carrier fluid without
being fixed to a substrate. In these techniques, the molecules are
analyzed as they move. While these latter techniques potentially
provide the same benefits of preserving the order and relative
separation of the landmarks, motion of the molecule complicates the
process of imaging the molecule, makes some landmarking techniques
difficult, and eliminates the possibility of preserving the
molecule for later additional or more complex analysis.
[0007] Ideally, when molecules are fixed to a substrate, the fixed
molecules should have sufficient separation so that molecules do
not overlap or cross. Points of overlap create image artifacts that
can severely hamper the analysis process.
[0008] It is typical to stain the fixed molecule with a fluorescent
material which distributes itself evenly along the molecule
allowing estimates of separation between landmarks (e.g., in
numbers of base pairs) to be gauged by total fluorescence rather
than strictly by length. Such fluorescence measurements work best
if the elongation of the molecule during straightening is not so
great as to decrease the fluorescence per length of the molecule to
a background level. Inadequate elongation of the molecule, however,
can make it difficult to identify the points cut by the restriction
enzymes, which desirably separate slightly under relaxation of the
elongated molecule to render the cuts visible.
[0009] Prior art techniques for elongating and fixing long chain
molecules can produce excessive overlap among molecules and
variation in molecule elongation.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method for straightening
and fixing polymeric molecules using well-controlled laminar flow
in a micro-channel. The laminar flow within a micro-channel allows
sufficient diffusion of the ends of the molecule so that they may
attach themselves to the wall of the micro-channel to be adsorbed
and fixed in their straightened configuration.
[0011] The present invention also provides an improved apparatus of
fabricating micro-channels suitable for this technique using an
elastic molding compound.
[0012] Additional embodiments of the present invention provide the
ability to sort molecular fragments by length and simple mechanisms
for producing the necessary controlled laminar flow.
[0013] Specifically, then, the present invention fixes and
straightens polymeric molecules using a channel sized to provide
laminar flow of a liquid along a channel length, the channel having
at least a first wall providing electrostatic attraction to the
polymeric molecule. A means is provided for passing the liquid and
polymeric molecule through the channel to straighten the polymeric
molecule by passage along the channel within the laminar flow and
allow absorption of the polymeric molecule to the first wall of the
channel in straightened form.
[0014] It is thus one object of the invention to provide an
improved method of straightening and fixing polymeric molecules.
The laminar flow may be controlled to provide more consistent
elongation to the molecules and improved separation of the
molecules with reduced overlap and better alignment.
[0015] The first wall of the channel may be transparent and, for
example, constructed of glass.
[0016] It is thus another object of the invention to provide an
optical mapping surface well suited for use with optical
microscopes.
[0017] The first wall may be treated to have a positive surface
charge of predetermined density.
[0018] Thus, it is another object of the invention to control the
electrostatic attraction between the polymeric molecule and the
optical mapping substrate for more precise control over the fixing
process.
[0019] The first wall may be detachable from the channel.
[0020] It is another object of the invention to provide an optical
mapping surface having improved accessibility and/or
reusability.
[0021] The channel may have at least one end that provides a funnel
section opening to a reservoir holding the liquid and polymeric
molecules.
[0022] It is thus another object of the invention to provide a
simple means for staging the polymeric molecules and one that
allows introduction of the polymeric molecules into the channel
with minimum breakage.
[0023] The means for passing the liquid and polymeric molecules
through the channel may, for example, be a pressure pump attached
to one end of the channel, for example, a syringe or other type of
pump, or a negative pressure pump attached to the other end of the
channel such as may draw the liquid through by pressure
differential, also, for example, being a syringe or other type of
pump. Alternatively, the means may be a reservoir acted on by a
force resulting from centrifugal acceleration of the channel and
reservoirs.
[0024] Thus, it is another object of the invention to provide a
variety of means of producing the necessary controlled laminar flow
in the channel.
[0025] The reservoir used when centrifugal acceleration provides
the movement of the liquid may be an end well extending
perpendicularly to the length of the channel and the apparatus may
further include a housing allowing the end well and channel to be
received by a centrifuge with the end well extending along a
principal axis of centrifugal acceleration, and the channel
extending substantially across the principal axis of centrifugal
acceleration.
[0026] Thus, it is another object of the invention to provide a
simple apparatus that makes use of a standard laboratory centrifuge
to produce the necessary flows and which thus may be inexpensive
and/or disposable.
[0027] The apparatus may include multiple end wells and multiple
micro-channels.
[0028] Thus, it is another object of the invention to allow
simultaneous parallel straightening and fixation of the same or
different polymeric molecules to occur to increase the throughput
of the analysis process.
[0029] The channel may include a region of varying cross-section to
promote a gradient in the laminar flow rate.
[0030] Thus, it is another object of the invention to provide for a
sorting of molecules by length, taking advantage of differences in
diffusion rate of the ends of the molecule as a function of
molecular length.
[0031] These particular objects and advantages may apply to only
some embodiments falling within the claims and thus do not define
the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a perspective view of one embodiment of the
present invention showing a micro-channel communicating between a
staging reservoir, holding polymeric molecules in a carrier liquid,
and a collecting reservoir, the micro-channel attached to the
reservoirs by funnel portions reducing shear and promoting laminar
flow in the micro-channel and showing the use of a syringe pump to
draw liquid through the micro-channel;
[0033] FIG. 2 is a cross-sectional view of the channel along lines
2--2 of FIG. 1 showing the increasing velocity of the laminar flow
in the micro-channel toward the center of the micro-channel and an
elongated DNA molecule centered in the micro-channel by the laminar
flow;
[0034] FIG. 3 is a fragmentary view similar to FIG. 2 showing: a
diffusion radius of one end of a polymeric molecule prior to
adsorption to a wall of the micro-channel; a polymeric molecule
having a trailing end attached to the wall of the micro-channel;
and a polymeric molecule having a leading end attached to the wall
of the micro-channel prior to adsorption of the entire length of
the polymeric molecule to the wall of the micro-channel;
[0035] FIG. 4 is a cross-sectional view of multiple micro-channels
during a first step of manufacturing the micro-channels in which a
mold is used with an elastic molding compound to form upper walls
of the micro-channels;
[0036] FIG. 5 is a figure similar to FIG. 4 showing removal of the
mold and attachment of the upper walls of the micro-channel to a
glass optical mapping surface in a second step of
manufacturing;
[0037] FIG. 6 is an elevational, cross-sectional view of an
alternative embodiment of the present invention in which
centrifugal acceleration acting on a fluid head in the staging
reservoir causes laminar flow in the micro-channel;
[0038] FIG. 7 is a plan view of the embodiment of FIG. 6 showing
multiple parallel micro-channels each with staging wells and
receiving wells;
[0039] FIG. 8 is a perspective view of the embodiments of FIGS. 6
and 7 as placed in a standard centrifuge cup, the latter in partial
cut-away;
[0040] FIG. 9 is a simplified diagram of rotation of centrifuge cup
of FIG. 8 showing the vectors of motion and centripetal
acceleration;
[0041] FIG. 10 is a plan view of an alternative micro-channel
design providing varying cross-sections and inversely varying flow
velocity such as may be used to sort polymeric molecules by size
along the length of the micro-channel; and
[0042] FIG. 11 is a flow diagram of the process of using the
present invention to straighten and fix polymeric molecules.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0043] Referring now to FIG. 1, the apparatus 10 of the present
invention provides a generally planar channel plate 12 into which a
longitudinally extending micro-channel 14 is formed, flanked by a
staging reservoir 16 and a collecting reservoir 18 positioned at
longitudinal ends of the channel plate 12.
[0044] Junctions between the longitudinal ends of the micro-channel
14 and staging reservoir 16 and collecting reservoir 18 are tapered
to create funnel sections with narrow ends attached to the
micro-channel 14 and wide ends attached to one of the staging
reservoir 16 or collecting reservoir 18. The funnel sections 20
provide a smooth transition of fluid from the staging reservoir 16
through the micro-channel 14 to the collecting reservoir 18 thereby
promoting laminar flow within the micro-channel 14 and reducing
breakage of polymeric molecules as will be described.
[0045] One common wall of the staging reservoir 16, the collecting
reservoir 18, and the micro-channel 14 is provided by an optical
mapping substrate 22 attached to the channel plate 12. The
substrate 22 thus encloses the staging reservoir 16, the collecting
reservoir 18, and the micro-channel 14. The substrate 22, for
example, may be a glass slide, treated as will be described
below
[0046] In the embodiment of FIG. 1, a sample introduction port 24
may be formed in the optical mapping substrate 22 at the staging
reservoir 16 to allow the introduction of polymeric molecules 36
and a carrier liquid 26 to the staging reservoir. In particular
embodiments, the sample introduction port 24 may be used for
pressure equalization when materials are drawn through the
micro-channel 14 or for the attachment of a pump to pressurize the
staging reservoir 16 to cause materials to flow through the
micro-channel 14.
[0047] Similarly, a sample extraction port 27 may be formed in the
optical mapping substrate 22 at the collecting reservoir 18 for
removal of material, pressure equalization, or as shown, the
attachment of pump 28 to draw the materials through the
micro-channel 14. Alternatively, the ports 24 and 27 may be formed
in the channel plate 12
[0048] In the embodiment of FIG. 1, the pump 28 is a syringe pump
providing precisely metered flow using an electromechanical
actuator and control system as is well understood in the art. The
syringe pump draws carrier liquid 26 and polymeric molecules 36
from staging reservoir 16 through the micro-channel 14 in the
collecting reservoir 18 at a controlled flow rate as may be set to
provide the desired laminar flow within the micro-channel 14.
[0049] Specifically, referring to FIG. 2, the laminar flow 30 of
carrier liquid 26 and polymeric molecules 36 within the
micro-channel is such as to provide flow 32 parallel to the
longitudinal walls of the micro-channel 14 with greatest flow
velocities toward the cross-sectional center of the micro-channel
14 thereby defining a flow velocity profile 34. The flow rate of
the pump 28 and the size of the micro-channel 14 is selected to
provide flow velocity profile 34 that promotes straightening of the
particular polymeric molecule 36 contained within the carrier
liquid 26 with the polymeric molecule 36 roughly centered within
the lumen of the micro-channel 14. These setting may be determined
empirically by visual observation of the polymeric molecules 36 at
different flow rates. Generally, laminar flow may be distinguished
from capillary flow in which the liquid is drawn along the surface
of the micro-channel 14 walls by a hydrophilicity of those walls
and where the center leading flow velocity profile 34 is not
obtained.
[0050] In a 50-micrometer wide micro-channel 14, for example, the
velocity of flow 32 may range from 15 to 70 micrometers per second
as measured across the lumen of the micro-channel 14.
[0051] In one embodiment, the cross-sectional width 38 of the
micro-channel 14 is 50 micrometers and is preferably less than 100
micrometers. More generally, it is believed that the width 38 will
be between one and one hundred times the straightened length 40 of
the polymeric molecule 36.
[0052] Referring now to FIG. 3, although the inventors do not wish
to be bound by a particular theory, it is believed that the ends 42
of the polymeric molecule 36 are more mobile than the remainder of
the polymeric molecule 36 and may be modeled as having an effective
diffusion radius 44 during the time the polymeric molecule 36 is in
transit in the micro-channel 14 and generally greater than the
polymeric molecule 36 as a whole. The average flow rate of the
carrier liquid 26 for the flow velocity profile 34 and the width 38
of the micro-channel 14 is thus adjusted so that this effective
diffusion radius 44 is equal to or greater than the width 38 of the
micro-channel 14. In this way, at some time during transit of the
polymeric molecule 36 within the micro-channel 14, contact by one
end 42 of a large number of the polymeric molecules 36 with the
substrate 22 can be expected. This contact will cause an
electrostatic bond between the substrate 22 and the end 42 of a
polymeric molecule 36.
[0053] Either the leading or the trailing ends 42 of the polymeric
molecule 36 may be the first to attach to the substrate 22. As
indicated by polymeric molecule 36', if the trailing end 42' of the
polymeric molecule 36'' is the first to contact the substrate 22 it
is believed that continued flow of the carrier liquid 26 pulls the
remainder of the polymeric molecule 36 against the substrate 22 to
be held there by electrostatic attraction in a straightened state.
Conversely, as indicated by polymeric molecule 36'', if the leading
end 42'' of the polymeric molecule 36'' is the first to contact the
substrate 22 it is believed that continued flow of the carrier
liquid 26 rolls the remainder of the polymeric molecule 36 over and
then draws it against the substrate 22 to be held there by
electrostatic attraction in a straightened state.
[0054] In order to promote and control attachment of the polymeric
molecule to the substrate 22, the substrate 22 may be treated to
establish a positive charge density on its surface contacting the
carrier liquid 26. For example, the surface may be derivative with
silage compounds, for example, those discussed in U.S. Pat. No.
5,720,928 hereby incorporated in its entirety by reference.
[0055] Whereas the micro-channels 14 and optionally the staging
reservoir 16 and collecting reservoir 18 of the apparatus 10 may be
constructed in silicon using conventional photolithographic
techniques, in a preferred embodiment of the present invention, the
micro-channels 14 (and optionally the staging reservoir 16 and
collecting reservoir 18) are constructed using a molded elastomeric
polymer.
[0056] Referring now to FIG. 4, in particular, a mold 45 providing
a planar substrate 46 with upstanding ridges 48 defining the volume
of the micro-channels 14 may be fabricated using conventional
photolithography in which a light sensitive photoresist is applied
to a silicon wafer that will form the mold 45. The photoresist is
hardened by selective optical exposure and the unhardened portions
washed away so that the photoresist provides a mask in the regions
of the upstanding ridges 48 (e.g., the regions of the
micro-channels 14 and the staging reservoir 16 and collecting
reservoir 18). The silicon wafer is then etched to a depth of 7 to
8 micrometers defining the height of the micro-channel 14.
[0057] Referring still to FIG. 4, an elastomeric polymer,
preferably poly(dimethylsiloxane) "PDMS") is then poured over this
mold 45 to create the channel plate 12. The PDMS channel plate 12
is then peeled from the mold 45 and exposed to oxygen plasma to
make it hydrophilic.
[0058] As shown in FIG. 5, the channel plate 12 may then be adhered
to the substrate 22 creating the micro-channels 14 and optionally
the staging reservoir 16 and collecting reservoir 18. The PDMS of
the channel plate 12 will naturally adhere to glass in a releasable
manner to produce a leak resistant seal. The seal is strong enough
to resist leakage of fluids filling the micro-channels for the
pressures used in this process yet weak enough to be reversible,
and thus make the channel plate 12 and substrates 22 reusable.
[0059] By treatment of the substrate 22, as described above, to
impress upon it a positive charge, and lack of treatment of channel
plate 12 or by a treatment that promotes a negative surface charge
on the channel plate 12 (?) preferential adsorption of the
polymeric molecules 36 by the substrate 22 may be promoted. Optical
mapping of the fixed polymeric molecules 36 may then be done
through the transparent glass substrate 22 by means of an inverted
microscope objective 50 such as a Zeiss Axiovert 135M such as is
readily commercially available. Before the optical mapping, the
polymeric molecule may be treated with fluorescent markers or
restriction enzymes as are understood in the art.
[0060] Alternatively, because the channel plate 12 is attached to
the substrate 22 releasable, the substrate 22 may be removed from
the channel plate 12 and the top surface of the substrate 22 may be
imaged. The removal of the channel plate 12 may also assist in
further treatment of the fixed polymeric molecules 36, for example,
with restriction enzymes and the like and the drying of these
molecules to further promote adhesion. The ability to separate the
channel plate 12 and substrate 22 allows one or both of these
elements to be reused if desired.
[0061] Referring now to FIG. 6, in an alternative embodiment to
that shown in FIG. 1, the height of the staging reservoir 16 and a
collecting reservoir 18 may be increased and ports 24 and 27
provided through the channel plate 12 opposite the substrate 22.
Upon initially filling staging reservoir 16 with carrier liquid 26
and polymeric molecules 36, a pressure head 52 is created being the
difference in liquid height in staging reservoir 16 and a
collecting reservoir 18. The small size of the micro-channel 14
limits flow from the staging reservoir 16 to the collecting
reservoir 18 under normal gravitational acceleration after limited
capillary flow.
[0062] Referring now to FIG. 8, the substrate 22 of the embodiment
of FIG. 6 may be attached to a weighted carrier 54 that fits within
the cup 56 of a standard swing bucket centrifuge 58 with the
channel plate 12 supported to be level with the top of the cup 56
and the staging reservoir 16 and collecting reservoir 18 extending
upward therefrom. The weighted carrier 54 is constructed so that
the combination of the channel plate 12, the substrate 22, and the
weighted carrier 54, when in position in the cup 56, have a center
of mass 57 below the pivot 55 about which the cup is free to
rotate.
[0063] When the centrifuge is started, as shown in FIG. 9, rotation
60 of the cups 56 swings them outward under the influence of a
radial centripetal acceleration 62 acting on the center of mass 57.
The acceleration promotes a downward force 64 shown in FIG. 6 on
the carrier liquid 26 sufficient to cause the desired laminar flow
through the micro-channel 14. By sizing the aperture of the
micro-channel 14, and controlling the initial pressure head 52, the
desired flow rate may be achieved.
[0064] Referring to FIGS. 6 and 7, a single channel plate 12 may
incorporate multiple staging reservoirs 16, collecting reservoirs
18 and intervening micro-channels 14. As the pressure head 52 drops
with flow through the micro-channel 14, the flow rate through the
micro-channel 14 will also decrease. Control of this rate of
decrease can be obtained by adjusting the relative diameter or
cross-sectional area of staging reservoir 16 compared to collecting
reservoir 18. For example, by making the collecting reservoirs 18
of bigger diameter than the staging reservoirs 16, the pressure
head 52 decreases more slowly. By making the diameter of the
reservoirs 16 and 18 large with respect to the flow rate or
concentrating the polymeric molecules in the bottom of the staging
reservoir 16, the molecules will pass through the micro-channel 14
only during the initial flow period providing more constant flow
and transit time of the polymeric molecules 36 through the
micro-channel 14.
[0065] Referring now to FIG. 10, in an alternative embodiment, the
micro-channel 14 may be given a varying cross-sectional area so
that for a given net flow rate 65 a series of different flow
velocities V1 through V3 will be created at different locations
along the micro-channel 14. It is believed that these varying flow
velocities may effect a spatial separation of polymeric molecules
36 according to their length. This length sorting may be desirable
to separate shorter polymeric molecules 36 from overlapping with
longer polymeric molecules or for analytic separation of polymeric
molecules 36 by length such as currently is done with
electrophoresis.
[0066] Referring now to FIG. 11, the present invention may be
incorporated as part of an optical mapping system. At a first step
70 of such a system, a solution, typically of water and polymeric
molecules, for example, DNA, is prepared by techniques well known
in the art. The polymeric molecules 36 may be treated with a
condensing agent such as spermine causing them to coil, thereby
reducing their damage during transfer to the apparatus 10 described
above.
[0067] At step 72, the water (which will act as the carrier liquid
26) and polymeric molecules 36 are inserted into the staging
reservoir 16. In the staging reservoir 16 they may be treated, for
example, with a saline solution to decondense the molecules over a
period, loosening their spermine-induced coiling. Once decondensed,
the carrier fluid 26 and polymeric molecules 36 flow through the
micro-channel 14 driven by a pump, centrifuge, or other method.
During the flow, polymeric molecules 36 attach to the substrate 22
in straightened configuration.
[0068] Additional treatment of the fixed polymeric molecules 36 may
be performed, as indicated by process block 74, by a variety of
methods known in the art including but not limited to tagging with
fluorescent materials or cutting by restriction enzymes. This step
may include staining the polymeric molecules 36 with a fluorescent
dye to provide accurate measurement of segments of the polymeric
molecules 36.
[0069] These treatments may be performed either by passing
additional liquids through the micro-channels 14 or by peeling back
the channel plate 12 to allow direct access to the polymeric
molecules 36 fixed to the substrate 22.
[0070] At process block 76, optical mapping of the fixed and
treated polymeric molecules 36 may be performed either through the
transparent optical mapping substrate 22 or by removing channel
plate 12. After optical mapping, the fixed polymeric molecules 36
may be stored.
[0071] The laminar fluid flow used in the present invention, in
contrast to radial or other capillary fluid flows is believed to
reduce the number of overlapping molecules. The controlled laminar
flow may also provide more consistent elongation or stretching of
the polymeric molecules 36.
[0072] It is specifically intended that the present invention not
be limited to the embodiments and illustrations contained herein,
but include modified forms of those embodiments including portions
of the embodiments and combinations of elements of different
embodiments as come within the scope of the following claims.
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